Insulin sensitivity in subjects with secondary hyperparathyroidism and the effect of a low serum 25-hydroxyvitamin D level on insulin sensitivity.

To investigate the relation between secondary hyperparathyroidism (SHPT) and insulin sensitivity, 15 subjects with SHPT (serum PTH >6.4 pmol/l, serum calcium <2.40 mmol/l, and normal serum creatinine) and 15 control subjects were investigated with an oral glucose tolerance test (OGTT) and a 3-h hyperglycemic clamp. Body composition was measured with dual-energy X-ray absorptiometry. No differences were found between the SHPT and control groups on any indices of glucose or insulin metabolism. However, when dividing the 30 subjects in the upper and lower halves according to serum 25-hydroxyvitamin D levels (<59 and >58 nmol/l), those in the lower half had significantly higher 2-h serum insulin value at the OGTT, significantly higher insulin secretion during the last hour of the clamp, and significantly lower insulin sensitivity index (ISI; glucose infusion rate/insulin secretion during the last hour of the clamp). In a multiple linear regression analysis correcting for age, gender, and body mass index (BMI), the serum 25-hydroxyvitamin D level was significantly and positively associated with the ISI. The amounts of total body and truncal fat were negatively and significantly associated with the ISI, whereas no association between measures of lean body mass were associated with insulin secretion or sensitivity.

Does rapid intraoperative parathyroid hormone analysis predict cure in patients undergoing surgery for primary hyperparathyroidism? A prospective study.

BACKGROUND AND AIMS: Preoperative 99mTc-sestamibi scintigraphy is used by many surgeons to identify the anatomical location of pathological parathyroid glands in patients undergoing surgical treatment for hyperparathyroidism. However, false negative results do occur. It has been suggested that intraoperative parathyroid hormone (PTH) analysis may enhance the possibility of performing successful focused, unilateral neck surgery in these patients. This study aimed to evaluate whether an adequate fall in intraoperative parathyroid hormone values predicts the removal of all hyperfunctioning parathyroid tissue and postoperative normocalcemia. MATERIAL AND METHODS: One hundred consecutive patients undergoing surgery for hyperparathyroidism had preoperative 99mTc-sestamibi scintigraphy and intraoperative parathyroid hormone (PTH) analysis. A fall in intraoperative PTH value by more than 50% of baseline value ended the procedure. This prospective study presents the clinical and biochemical results. RESULTS: The overall sensitivity of the 99mTc-sestamib scintigraphy was 88% and for single adenomas 95%. The scintigraphy failed to detect the correct pathology in all cases with multiglandular disease (7 patients). A fall in intraoperative PTH value by more than 50% of baseline value was achieved in all patients. The combination of intraoperative PTH analysis and 99mTc-sestamibi scintigraphy enabled us to limit the operation to a focused, unilateral operation in 87 of the 100 patients. All patients were normocalcemic postoperatively. CONCLUSIONS: A fall in intraoperative PTH value more than 50 % of baseline value seems to predict postoperative normocalcemia and the removal of all hyperfunctioning parathyroid tissue. Bilateral neck exploration is avoided in the majority of patients.

Serum parathyroid hormone (PTH) levels in smokers and non-smokers. The fifth Tromsø study.

OBJECTIVE: Smoking is associated with reduced bone density and calcium absorption, and reduced serum levels of vitamin D. A compensatory increase in serum parathyroid hormone (PTH) would therefore be expected as a result of an altered calcium balance. However, reports on PTH levels in smokers are conflicting. As serum PTH levels give important information on the calcium balance, the PTH levels in smokers are of interest. SUBJECTS AND METHODS: In the fifth Tromsø study, smoking status was recorded and serum PTH measured in 7896 subjects. Intakes of calcium and vitamin D were evaluated with a food-frequency questionnaire. In a follow-up study on 205 subjects, serum 25-hydroxyvitamin D, calcium absorption, and renal excretion of calcium were measured in addition. RESULTS: The serum PTH levels were significantly lower in smokers than non-smokers (3.1+/-1.4 vs 3.6+/-1.9 pmol/l in males; 3.1+/-1.5 vs 3.6+/-1.8 pmol/l in females (P < 0.001) after correcting for confounding variables, linear regression). In the smokers, there was no association between number of cigarettes smoked and serum PTH. One year after quitting smoking, serum PTH levels were similar to those of people who had never smoked. The smokers had significantly lower intake of vitamin D, lower serum levels of 25-hydroxyvitamin D and lower calcium absorption. The intake of calcium and the renal excretion of calcium were similar to that in non-smokers. CONCLUSIONS: Smokers have lower serum PTH levels than non-smokers. This cannot be explained by the predictors of serum PTH measured in our study.

Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo, Norway: the Oslo Immigrant Health Study.

OBJECTIVE: To study the prevalence of vitamin D deficiency and to identify possible predictors of vitamin D deficiency in five main immigrant groups in Oslo. DESIGN: Cross-sectional, population-based. SETTING: City of Oslo. SUBJECTS: In total, 491 men and 509 women with native countries Turkey, Sri Lanka, Iran, Pakistan and Vietnam living in the county of Oslo. RESULTS: Median serum 25(OH)D level (s-25(OH)D) was 28 nmol/l, ranging from 21 nmol/l in women born in Pakistan to 40 nmol/l in men born in Vietnam. Overall prevalence of vitamin D deficiency defined as s-25(OH)D<25 nmol/l was 37.2%, ranging from 8.5% in men born in Vietnam to 64.9% in women born in Pakistan. s-25(OH)D did not vary significantly with age. s-25(OH)D was higher in blood samples drawn in June compared to samples obtained in April, but not significantly for women. Reported use of fatty fish and cod liver oil supplements showed a strong positive association with s-25(OH)D in all groups. Education length was positively associated with s-25(OH)D in women, whereas body mass index (BMI) was inversely associated with s-25(OH)D in women. These two variables were not related to vitamin D deficiency in men. CONCLUSIONS: There is widespread vitamin D deficiency in both men and women born in Turkey, Sri Lanka, Iran, Pakistan and Vietnam residing in Oslo. The prevalence of vitamin D deficiency is higher in women than in men, and it is higher in those born in Pakistan and lower in those born in Vietnam compared to the other ethnic groups. Fatty fish intake and cod liver oil supplements are important determinant factors of vitamin D status in the groups studied. BMI and education length are also important predictors in women.

Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein.

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.

Effects of sex steroids on prolactin secreting rat pituitary cells in culture.

A clonal strain of rat pituitary tumor cells (GH3) was used to study the effects of different sex steroids on the production of prolactin (PRL). Hormone production was measured by radioimmunoassay and expressed as the amount of hormone which accumulated in the medium of monolayer cultures during 24 h. The stimulatory effect of 17beta-estradiol (10(-11)M-10(-6)M) on PRL production was significant after 4 days and the maximum effect (300% of control cultures) was observed at 10(-8)M after 10 days of treatment. After removal of added 17beta-estradiol, the production of PRL returned to control levels in 5 days. Progesterone (10(-11)M-10(-6)M) caused a dose-related decrease in PRL production reaching 60% of control values at 10(-6)M. Testerone (10(-6)M) stimulated the production of PRL (130% of controls), whereas 5alpha-dihydrotestosterone (10(-6)M) had a small effect (107% of controls) which was not always reproducible. None of the sex steroids affected cell growth. Progesterone (10(-6)M) inhibited the stimulatory effect of 17beta-estradiol (10(-8)M) on PRL production. The effect of 17beta-estradiol (10(-8)M) and thyrotropin releasing hormone (TRH) (3 X 10(-7) M) was addititive, while no additional stimulatory effect was observed when 17beta-estradiol (10(-8)M) was combined with testosterone (10(-6)M). If the properties of the GH3 cells are analogous to those of normal lactotropes, the sex steroids may alter PRL production at the pituitary level, an influence that may be further modulated by TRH.

The mechanism of 17 beta-estradiol uptake into prolactin-producing rat pituitary cells (GH3 cells) in culture.

Estrogens stimulate PRL synthesis in cultured GH3 cells, which are clonal strains derived from the rat pituitary gland. This model system was used to study the mechanism by which 17 beta-estradiol (E2) enters target cells. The cellular uptake of [3H]E2 was rapid at 37 C and reached half-maximal values within 10-15 sec. Maximal uptake was observed in less than 2 min at 37 C. The initial rates of E2 uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]E2 in intact cells and the binding to cytosol studied at different temperatures resulted in linear Arrhenius plots, and the energies of activation were 39.0 and 33.5 kJ mol-1 degree-1, respectively. Purified GH3 cells membrane fractions, which showed specific binding sites for [3H]TRH, displayed the same maximal binding of [3H]E2 in the absence or presence of cold hormone. The amount of membrane-associated [3H]E2 increased linearly with temperature and extra-cellular hormone concentration. The effect of temperature on binding of E2 to membrane fractions occurred gradually without phase transitions and was not saturable. We suggest that the mechanism by which E2 is taken up by target cells at physiological temperature involves instantaneous dissolution in the cell membrane from where it diffuses passively into the cell. E2 binds thereafter to specific receptors in an energy-dependent step.

Androgen receptors in the anterior pituitary and central nervous system of the androgen "insensitive" (Tfm) rat: correlation between receptor binding and effects of androgens on gonadotropin secretion.

The cytosol fractions of the anterior pituitary, hypothalamus, preoptic area and brain cortex of androgen "insensitive" (Tfm) rats possess androgen receptors. However, in the Tfm rats the androgen binding per mg protein was only 10-15% of that in the corresponding normal littermates (Nl). The physicochemical properties of the androgen receptors in the anterior pituitary of the Tfm rat were indistinguishable from those of the normal rat. Thus, no distinctive differences were observed with regard to electrophoretic mobility in 3.25% polyacrylamide gels, isoelectric point (pI=5.8), binding affinity (KD=1.5 X 10(-9)M), temperature stability, sulfhydryl dependence and steroid specificity. It is, therefore, likely that the very low androgen binding capacity by the anterior pituitary and the central nervous system is due to an extreme reduction in the receptor number rather than to the presence of abnormal receptors. Since in the Tfm animals the androgen receptor number is reduced by 85-90%, it is to be expected that very high doses of androgens would be required to achieve hormonal effects. In fact, low doses of 5alpha-dihydrotestosterone propionate (50 mug/100 g body weight) given sc daily for 12 days had no effect on serum levels of LH and FSH. However, very high doses (2 mg/100 g body weight) of testosterone propionate and 5alpha-dihydrotestosterone propionate, which maintained circulating androgen levels above 20 ng/ml, significantly reduced serum gonadotropin levels in castrated Tfm rats. In normal littermates both low and high doses of the androgens suppressed gonadotropin secretion to low levels. These findings strongly indicate that androgen receptors are essential to androgen action on the anterior pituitary and central nervous system in the rat. The serum levels of testosterone (7.7+/-0.15 (SE) ng/ml) and 5alpha-dihydrotestosterone (0.37+/-0.06 ng/ml) were significantly higher in intact Tfm rats than in normal littermates (2.6+/-0.03 and less than 0.1 ng/ml, respectively). The failure of the elevated concentrations of serum androgens to reduce the high serum levels of LH and FSH in intact Tfm rats is most likely due to the extreme reduction of the androgen receptor number and the consequent insufficient hypothalamic and/or pituitary response to androgens.

1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor number and increasing the level of the AC inhibitory G-protein Gi-2 alpha in FRTL-5 cells.

Early postmenopausal bone loss is not associated with a decrease in circulating levels of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, or vitamin D-binding protein.

To study changes in the rate of bone loss before and after the menopause, 19 normal premenopausal women of the same age were followed with annual measurements of appendicular bone mass for 8 yr. Their serum estrone, estradiol, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and vitamin D-binding protein levels also were measured annually. The serum estrogen levels declined before the menopause, and the rate of bone loss more than doubled at the time of the menopause. There were no significant changes in the serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin LD or vitamin D-binding protein levels from the pre- to the postmenopausal period. We conclude that changes in vitamin D metabolite levels are not associated with early postmenopausal bone loss.

Plasma gastrointestinal hormones during spontaneous and induced menstrual cycles.

Plasma levels of secretin, vasoactive intestinal polypeptide (VIP), somatostatin (SRIH), motilin, and/or pancreatic polypeptide, as well as serum estradiol, progesterone, PRL, LH, FSH, and/or GH were measured during the follicular phase, midcycle, and luteal phase of a spontaneous menstrual cycle in eight women and during ovarian stimulation with clomiphene citrate/human menopausal gonadotropin and hCG for in vitro fertilization in nine women. Plasma SRIH concentrations were significantly (P less than 0.02) higher in the luteal phase of spontaneous menstrual cycles than in follicular phase and midcycle. Serum GH levels, however, did not change. Plasma motilin concentrations also were higher in the luteal phase than at mid-cycle (P less than 0.04). Plasma secretin, VIP, and pancreatic polypeptide concentrations did not change during the cycle. Throughout the spontaneous menstrual cycle we found significant positive correlations between plasma SRIH and serum progesterone (P less than 0.007; r = 0.5869), plasma motilin and serum progesterone (P less than 0.02; r = 0.5331), plasma secretin and serum estradiol (P less than 0.04; r = 0.4711), and plasma secretin and serum PRL (P less than 0.02; r = 0.5507). During ovarian stimulation both plasma secretin and VIP gradually increased to a peak on cycle days 0 and 1, respectively (day 0 = the day of hCG injection), whereas plasma SRIH did not change. Serum estradiol and PRL increased significantly, and both peaked on cycle day 1. During ovarian stimulation plasma secretin correlated significantly with serum estradiol (P less than 0.00001; r = 0.9333), serum PRL (P less than 0.03; r = 0.6521), and plasma VIP (P less than 0.03; r = 0.6534). In addition, plasma VIP and serum PRL both correlated significantly with serum estradiol (P less than 0.05; r = 0.6024 and P less than 0.04; r = 0.6384, respectively). These results indicate 1) a possible effect of progesterone on the release of SRIH and motilin during the spontaneous menstrual cycle; 2) the unaltered serum GH concentrations in the luteal phase of the spontaneous menstrual cycle despite elevated plasma SRIH levels are probably due to a stimulatory effect of both progesterone and motilin on GH release; and 3) the increase in plasma secretin and VIP concentrations during ovarian stimulation is probably secondary to the concomitant increase in serum estradiol and/or PRL. We suggest that estradiol and/or PRL beyond a certain threshold level stimulate the release of secretin, and possibly also VIP, into plasma.

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis.

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.

24,25-dihydroxy vitamin D3 treatment inhibits parathyroid-stimulated adenylate cyclase in iliac crest biopsies from uremic patients.

Renal osteodystrophy with increased bone resorption is a major clinical problem in patients with chronic renal failure. Previous reports have shown that treatment with 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) may result in decreased bone resorption. The present study addresses basic mechanisms for the action of 24,25(OH)2D3 in bone of patients with elevated serum parathyroid hormone (PTH) levels due to chronic renal disease. Twenty-four patients 56 +/- 17 years old (mean +/- SE) with chronic kidney disease in the predialytic state (serum creatinine > 150 mumol/l) and elevated serum midregion PTH > 1.2 micrograms/l were randomly assigned to oral treatment with either 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) (0.25-0.50 microgram/day), 24,25(OH)2D3 (daily dose of 15 micrograms), or a combination of the two vitamin D3 analogs. The control group received calcium carbonate (maximal dosage of 1 g x 3). Selected variables in serum and urine as well as hormone sensitive adenylate cyclase (AC) in iliac crest biopsies were assessed before treatment and during follow-up after two and six months. Serum levels of 1,25(OH)2D3 and 24,25(OH)2D3 were significantly (P < 0.05) increased after two and six months in the respective treatment groups. Net bone PTH-enhanced AC (PTH-AC) fell abruptly (P < 0.01) after two months of treatment and was nearly abolished (P < 0.01) after six months with 24,25(OH)2D3 given alone or in combination with 1,25(OH)2D3. An inverse relationship (r = -0.57, P < 0.05, n = 48) between net PTH-AC in bone and serum levels of 24,25(OH)2D3 was demonstrated. In all groups, serum total calcium (s-Ca) was maintained within normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen replacement and quality of life in patients treated for bilateral testicular cancer.

Gonadal hormones and quality of life (QL) were assessed in bilaterally orchiectomized patients with testicular cancer who received intramuscular androgen replacement (ARP). 43 patients were to have serum analyses of testosterone LH, FSH and SHBG, preferably performed at the end of the interval between two intramuscular injections. They also completed a QL questionnaire consisting of the EORTC QLQ-C30, GHQ-28, IES and PAIS (sexuality). 17 of 31 evaluable patients had subnormal testosterone levels, and 9 highly elevated LH. Blood levels indicating hypogonadism were more often observed in the 25 patients whose ARP was scheduled at > or = 3 week intervals than in the 18 patients with < or = 2 weeks between ARP injections. A total of 11 patients reported hot flushes. The patients' QL was similar to that of a control group. However, 8 (20%) patients were 'cases' according to GHQ-28/IES, independent of their hormone levels. Current standard intramuscular ARP is not optimal in approximately 1/3 of the patients who have undergone bilateral orchiectomy for testicular cancer, particularly if scheduled at > or = 3 week intervals. Schedules for ARP have to be improved. In spite of intermittent hypogonadism most patients are psychosocially and sexually well adjusted to their health situation.

Human IgG subclass-specific rabbit antisera suitable for immunoprecipitation in gel, ELISA and multilayer haemagglutination techniques.

Antisera specific for human IgG subclasses were raised in rabbits by tolerance induction at birth followed by active immunization at 8 weeks of age. This procedure gave better antisera compared to tolerance/immunization of adult grown animals. Many animals gave antisera with subclass-specific precipitating activity without the need to use absorption procedures. However, for sensitive techniques such as ELISA and multilayer haemagglutination it was necessary to absorb the antisera to make them subclass-specific. Subclass specificity in ELISA and haemagglutination was verified by inhibition. We believe this is the first description of a procedure to prepare antisera specific for human IgG subclasses suitable for use in immunoprecipitation, ELISA and haemagglutination techniques.

Perfusion system for studying peptide secretion from endocrine cells with high time resolution.

To study the secretion from endocrine cells in culture, we have developed a cell column perfusion system with a time resolution of 4 s. The core of the system is a perfusion chamber with a cell-supporting matrix of monosized polystyrene beads. The particles are solid and can withstand a high pressure gradient without deformation. The minimum amount of cell material required to obtain detectable levels of secretory products is a function of the assay sensitivity, perfusion flow, fraction volume and time resolution. The volume of the perfusion chamber is therefore adjustable to satisfy varying demands of minimum cell number. The general flow characteristics of the system were characterized using radiolabeled substances of various molecular sizes. Using the system in secretory studies of rat pituitary tumor (GH4C1) cells, we have identified differences in secretion profiles that may be related to the kinetics of the different transmembrane and intracellular mechanisms involved.

Mouse trophoblast cells are constitutive producers of thromboplastin (factor III) in vitro.

Trophoblasts from murine placenta synthesize thromboplastin in the absence of inducing agents and a functional complement system, nor is the rate or level of synthesis enhanced by inducers. A serum factor which is destroyed/removed by addition of oxalate and subsequent dialysis appears to enhance the ability of trophoblasts to synthesize thromboplastin.

Phorbol esters and thyroliberin have distinct actions regarding stimulation of prolactin secretion and activation of adenylate cyclase in rat pituitary tumour cells (GH4C1 cells).

The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) enhances the effects of TRH on phase II of prolactin secretion as well as on hormone synthesis at both low and high TPA receptor occupancy. Furthermore TPA, but not the biologically inactive substance 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), stimulates the particulate bound adenylate cyclase with a time course paralleling that of TRH activation. However, the combined additions of TRH and TPA activate this cyclase in an additive manner while the Gpp(NH)p- and the forskolin-sensitive enzyme are unaffected by TPA addition. Polymyxin B, which inhibits protein kinase C, abolishes activation of adenylate cyclase by TPA without interfering with the stimulatory action of TRH. Also, when phosphatase activity is preferentially inhibited by pretreatment of the cells with sodium vanadate, the TRH-sensitive cyclase is unaltered, while TPA activation is obliterated. Maximal stimulation of adenylate cyclase by cholera toxin pretreatment, obliterated the actions of TRH and TPA. Cells pretreated with pertussis toxin retained their TRH-sensitive cyclase, however, TPA-responsiveness was lost. We therefore suggest that the action of TPA as it relates to activation of adenylate cyclase, is probably mediated via the Gi component of the adenylate cyclase complex, while TRH stimulates the enzyme via the classical pathway involving the stimulatory GTP binding protein (Gs).

Calcitriol attenuates the thyrotropin-releasing hormone-stimulated inositol phosphate production in clonal rat pituitary (GH4C1) cells.

Three days pretreatment of the prolactin (PRL) secreting GH4C1 cells with 10 nM calcitriol attenuated both the basal and thyrotropin-releasing hormone (TRH)-stimulated (1 microM, 5 s) inositol trisphosphate (IP3) production by 30 and 26%, respectively. The effect was detectable at 10 nM (basal) and 1 pM (TRH-stimulated), and maximal at 1 microM (basal) and 10 nM (TRH), respectively. Calcitriol was at least 100 times more potent than calcidiol and 24-hydroxycalcidiol, and the effect was reversible upon cessation of pretreatment. Calcitriol pretreatment (1 microM, 5 days) also decreased the levels of phosphatidyl-inositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by 23, 55 and 32%, respectively. GTP gamma S-stimulated (100 microM, 30 s) IP3 production was decreased by 45% after calcitriol pretreatment (10 nM, 5 days). Pertussis toxin (1 nM, 4 h) attenuated both the basal and TRH-stimulated IP3 production, but this effect was omitted by calcitriol pretreatment. Thus, calcitriol specifically attenuates both the basal and TRH-stimulated inositol phosphate production in GH4C1 cells. The mechanism, at least partly, involves decreased availability of phosphoinositides for phospholipase C. Calcitriol regulation of a pertussis toxin-sensitive G-protein might also play some role.

Receptors for 17beta-estradiol in prolactin-secreting rat pituitary cells.

Estrogens stimulate prolactin (PRL) synthesis by GH3 cells, a clonal strain of rat pituitary cells grown in culture. At 4 degrees C the binding of [3H]17 beta-estradiol to monolayer cultures of GH3 cells was specific and of limited capacity, with half-maximal and maximal binding after 1--2 h and 12 h, respectively. Scatchard analysis showed one single class of binding sites with Kd = 3.1 X 10(-10) M and n = 309 X 10(-15) mol 17 beta-estradiol/mg cell protein, calculated to give approx. 25,000 binding sites per cell. At 4 degrees C less than 10% of the specifically bound [3H]17 beta-estradiol was found in the nuclear fraction. When the incubation temperature was raised to 37 degrees C, the amount of radioactivity in the nucleus increased to 25% within 30 min with a corresponding reduction in the cytoplasm. The cytosol fractions from monolayer cultures as well as from tumors of GH3 cells contained specific 17 beta-estradiol binding proteins, having a sedimentation constant close to 8S in a salt-free buffer and 4S in the presence of 0.5 M KCl. scatchard analysis showed one single class of binding sites with Kd = 3.6 X 10(-10) M and n = 258 X 10(-15) mol 17 beta-estradiol/mg cytosol protein (GH3 tumor tissue). Thus, GH3 cells grown in culture and in the intact animal have similar binding characteristics as judged from the data for binding affinity, capacity and specificity. After the in vivo administration of [3H]17 beta-estradiol to GH3 tumor-bearing rats, radioactivity could be extracted (0.5 M KCl) from purified nuclei bound to 4.5S macromolecules. We suggest that the action of 17 beta-estradiol on GH3 cells involves an initial binding of the steroid to specific receptors in the cytoplasm, followed by transport of a fraction of the hormone-receptor complexes to the nucleus involving a temperature-sensitive step.